Solid phase synthesis has been known for some time for use in the preparation of peptides and other oligomers such as nucleotides. Such solid phase synthesis makes use of an insoluble resin support for a growing oligomer. A sequence of subunits, destined to comprise a desired polymer, are reacted together in sequence on the support. A terminal amino acid, nucleotide or other residue is attached to the solid support in an initial reaction, either directly or through a keying agent. The terminal residue is reacted, in sequence, with a series of further residues such as amino acids or blocked amino acid moieties to yield a growing oligomer attached to the solid support through the terminal residue. At each stage in the synthetic scheme, unreacted reactant materials are washed out or otherwise removed from contact with the solid phase. The cycle is continued with a pre-selected sequence of residues until the desired polymer has been completely synthesized, but remains attached to the solid support. The polymer is then cleaved from the solid support and purified for use. The foregoing general synthetic scheme was developed by R. B. Merrifield for use in the preparation of certain peptides. It has also been adapted for the preparation of oligo and poly nucleotides.
These schemes are known to persons having ordinary skill in the art. See Merrifield's Nobel Prize Lecture "Solid Phase Synthesis", Science, Vol. 232, pp. 341-347 (1986), incorporated herein by reference. They have also been widely reviewed. See, for example, "Solid-Phase Peptide Synthesis", Doscher, in Methods in Enzymology, Volume 47, Enzyme Structure Part E, pp. 578-617, Academic Press (1977); "Solid-Phase Peptide Synthesis, Erickson and Merrifield in The Proteins, Third Edition, Volume 2, at pp. 255-527, Neurath et al, ed., Academic Press (1976); Stewart, "Solid Phase Peptide and Protein Synthesis" in Solid Phase Biochemistry, Scouten, ed., Chapter 10, pp. 507-534 Wiley (1983); and Solid Phase Peptide Synthesis, 2nd ed., Stewart & Young, Pierce Chem. Co. (1984) which are incorporated herein by reference.
It is also known to prepare polynucleotides from nucleotide or blocked nucleotide moieties through solid phase synthesis. Such methods are analogous in concept to the synthetic methods for polypeptides and are, similarly, known to persons of ordinary skill in the art. See, for example, Ikehara, Ohtsuka and Markham, Adv. Carbohydrate Chem. Biochem., 36: 135-213 (1979); "Solid Phase Synthesis and Biological Applications of Polydeoxyribonucleotides", Wallace et al. in Solid Phase Biochemistry, Scouten ed., ibid., pp. 661-663. The foregoing are incorporated herein by reference.
A major disadvantage of solid phase synthetic methods for the preparation of oligomeric materials such as peptides and nucleotides results from the fact that the reactions involved in the scheme are imperfect; no reaction proceeds to 100% completion. As each new subunit is added to the growing oligomeric chain a small, but measurable, proportion of the desired reactions fails to take place. The result of this is a series of peptides, nucleotides, or other oligomers having deletions in their sequence. This problem is particularly acute for the addition of valine and aspartic acid amino acid moieties to peptide chains. The result of the foregoing imperfection in the synthetic scheme is that as desired chain length increases, the effective yield of desired product decreases drastically, since increased chances for deletion occur. Similar considerations attend other types of unwanted reactions, such as those resulting from imperfect blocking, side reactions, and the like.
Of equal, if not greater, significance, is the fact that the increasing numbers of undesired polymeric species which result from the failed individual reactions produce grave difficulties in purification. Thus, for example, if a polypeptide is desired having 100 amino acid residues, it can be seen that there may be as many as 99 separate peptides having one deleted amino acid residue and an even greater possible number of undesired polymers having two or more deleted residues, side reaction products and the like. While each of the undesired peptides may comprise but a small proportion of the overall mole percentage of product, taken in sum the impurities attain a substantial percentage. Moreover, since their structure is very similar to that of the desired peptide, the difficulties of purifying the desired peptide from the melange becomes formidable. Similar difficulties attend the preparation of polynucleotides from their constituent nucleic acids or nucleotides.
The foregoing difficulties are exacerbated by the fact that in traditional solid state synthetic schemes, the resulting oligomer, such as a peptide or polynucleotide, is not generally purified until the synthetic scheme is completed and the product mixture removed from the solid support. Some workers, including Merrifield himself, have attempted to overcome this difficulty in the preparation of polypeptides by attaching the terminal amino acid group to the solid support with a selective, cleavable group such as one which is photolabile. After elaboration of 15 to 20 amino acid residues, the growing polypeptide chains are cleaved from the solid support, subjected to purification, and only the desired intermediate products caused to become reattached to a solid support for further elaboration of structure. Theoretically, this technique could be repeated several times to prepare large polypeptides. In actuality, however, the selective cleavage and reattachment of growing polypeptide chains from and to solid supports proceeds with only a low yield. Thus, the overall yield of polypeptide in accordance with these schemes is poor; the yield decreases with increasing peptide size. Similar difficulties are known for polynucleotides.
Another technique which has received wide acceptance in the preparation of peptides enjoys the benefit of simplicity. In accordance with this technique, each peptide bond formation reaction is repeated twice, and even more often for particularly difficult amino acids, in order to improve the overall yield of each step. While deletion of amino acid residues is substantially diminished through the use of this technique, it is not eliminated and is slow, time consuming, and costly.
Yet another approach has been to remove aliquots of the growing, solid supported, peptide or other oligomeric chain, to test the same for purity through a variety of characterization protocols and to take appropriate chemical steps to ensure high completeness in the reaction scheme. This approach is also laborious, time consuming and costly.
Yet another approach has been followed which approximates the concept of parallel synthesis known to organic chemists. Thus, some workers have prepared moderately sized peptides, i.e. about 15 amino acid units, which can successfully be purified without excessive difficulty. These segments are then assembled through traditional wet or solid-phase chemistry into the desired, larger peptide. While attractive in theory, this technique has not been shown to be particularly useful. Moderately sized polypeptides may attain a variety of conformations, certain of which are believed to inhibit the effective reaction of those segments with the desired, adjacent segments in the overall polypeptide. The result of this is generally poor yields. Similar attempts have also been made in nucleic acid synthesis without notable success.
A further approach has been to terminate the length of peptides generally by acetylation. While limiting the number of side reactions, the scheme does not provide a good solution for large molecules.
Evidence of the long-felt need for improved methods to attain higher purity in solid-state synthesis of proteins is reflected inter alia, in "Affinity and Carrier-Mediated Peptide Purification", Wilchek and Miron, Peptides, Structure and Function, (1979), E. Gross and J. Meienhofer, Eds., Pierce Chem. Co., Rockford, Ill., pp. 49-57; and "Solid Phase Peptide Synthesis", Merrifield et al, ibid., pp. 29-47. Further pressure to attain solutions to the purification problems attendant to solid phase synthesis of proteins has arisen from the competing technology of genetic engineering. See "Proteins to Order", Tucker, High Technology, Dec. 1985; pp. 26-34; and "Protein Engineering, Biotechnology's New Wave", ASM News, pp. 566-568 (1985). Previously, however, no solution has been forthcoming.